To address the challenge of producting stable polypeptide compositions, proteins are typically prepared in solid form to provide an acceptable shelf life. A standard method for preparing solid form protein compositions is lyophilization (freeze-drying) but this process creates stresses which can denature proteins to varying degrees (Wang, W. (2000) International Journal of Pharmaceutics 203; 1-60). Few biological reaction compounds are stable in solubilized form for any length of time and this is especially true for storage at room temperature. Consequently, numerous studies have been performed to evaluate possibilities to enhance the storage capabilities of biological reaction compounds in dry form. It is generally accepted that it will be necessary to use at least one stabilizing additive in order to assure the biological activity of e.g. a polymerase upon re-solubilization.
WO 2008/36544 describes the use of so-called filler materials in order to provide dried compositions, the filler materials are e.g. carbohydrates such as FICOLL, sucrose, glucose, trehalose, melezitose, DEXTRAN or mannitol, proteins such BSA, gelatin or collagen and polymers such as PEG or polyvinyl pyrrolidone (PVP). Glass-forming filler materials for stabilizing biological reagents are further described in U.S. Pat. Nos. 5,098,893, 5,200,399 and 5,240,843. The filler material FICOLL is a copolymer disclosed in U.S. Pat. No. 3,300,474. The methods of drying the liquid reaction mixtures are most of the time very complex in nature and therefore, the drying procedures are demanding and expensive.
Freeze-drying (U.S. Pat. No. 5,593,824) or vacuum drying (U.S. Pat. No. 5,565,318) is used for drying the biological materials in a carbohydrate polymer matrix. Lyophilization or freeze-drying is a well established technique towards storage of proteins that is disclosed in many state of the art documents (e.g. Passot, S., et al., Pharmaceutical Development and Technology 12 (2007) 543-553; Carpenter, J. F., et al., Pharmaceutical Research 14 (8) (1997) 969-975; Schwegman, J. J., et al., Pharmaceutical Development and Technology 10 (2005) 151-173).
A selection of drying conditions for different reaction mixtures for sequencing applications comprising genetic modifications of the Taq polymerase are described in U.S. Pat. No. 7,407,747. Drying procedures used are freeze-drying, speedvac without additional heat, speedvac with additional heat and air drying at room temperature. The reaction mixtures within this patent were tested with respect to a variety of cryoprotectants such as trehalose, sucrose, glucose and trimethylamine-N-oxide (TMANO). Moreover, experiments were also performed without cryoprotectants at all, but no data was disclosed concerning the stability of those reaction mixtures with time. A good stability for as long as 8 weeks was reported only for reaction mixtures comprising trehalose and bovine serum albumin (BSA).
Moreover, U.S. Pat. No. 7,407,747 discloses experiments with the polymerase in different sequencing mixtures where each sequencing mixture comprises different compositions of buffer solution, nucleotide triphosphates, and nucleotides with fluorescence label and primers. However, there is no disclosure if a polymerase in mixtures for real-time PCR amplifications, namely mixtures comprising buffer solution, nucleotides triphosphates, primers and detection probes, may be dried and stored without affecting the PCR activity of the polymerase.
U.S. Pat. No. 8,652,811 discloses a method to dry a Taq DNA polymerase within a real-time PCR mixture, whereas the obtained dry composition can be stored without affecting the PCR performance of the Taq DNA polymerase.